The European Pharmacopoeia describes a liquid chromatography (LC) method for the quantification of sulindac, using a quaternary mobile phase including chloroform and with a rather long run time. In the ... [more ▼]

The European Pharmacopoeia describes a liquid chromatography (LC) method for the quantification of sulindac, using a quaternary mobile phase including chloroform and with a rather long run time. In the present study, a new method using a short sub-2μm column, which can be used on a classical HPLC system, was developed. The new LC conditions (without chloroform) were optimised by means of a new methodology based on design of experiments in order to obtain an optimal separation. Four factors were studied: the duration of the initial isocratic step, the percentage of organic modifier at the beginning of the gradient, the percentage of organic modifier at the end of the gradient and the gradient time. The optimal condition allows the separation of sulindac and of its 3 related impurities in six minutes instead of 18 min. Finally, the method was successfully validated using an accuracy profile approach in order to demonstrate its ability to accurately quantify these compounds. [less ▲]

To investigate the encapsulation of Print 3G, a peptidic agent that could reduce the angiogenic development of breast tumors, pegylated liposomes used as intravenous vectors were studied and characterized ... [more ▼]

To investigate the encapsulation of Print 3G, a peptidic agent that could reduce the angiogenic development of breast tumors, pegylated liposomes used as intravenous vectors were studied and characterized. Recently, the path of liposomes has been explored with success to improve the pharmacological properties of peptidic drugs and to stabilize them. In this study, loaded unilamellar vesicles composed of SPC:CHOL:mPEG2000-DSPE (47:47:6) were prepared by the hydration of lipid film technique. An HPLC method was developed and validated for the determination of Print 3G to calculate its encapsulation efficiency. Observed Print 3G adsorption on different materials employed during liposome preparation (such as glass beads, tubing, and connections for extrusion) led to the modification of the manufacturing method. The freeze-thawing technique was used to enhance the amount of Print 3G encapsulated into blank liposomes prepared using the hydration of lipid film procedure. Many factors may influence peptide entrapment, namely the number of freeze-thawing cycles, the lipid concentration, the peptide concentration, and the mixing time. Consequently, a design of experiments was performed to obtain the best encapsulation efficiency while minimizing the number of experiments. The lipid concentration and the number of freeze-thawing cycles were identified as the positive factors influencing the encapsulation. As a result of the optimization, an optimum was found and encapsulation efficiencies were improved from around 30% to 63%. Liposome integrity was evaluated by photon correlation spectroscopy and freeze-fracture electron microscopy to ensure that the selected formulation possesses the required properties to be a potential candidate for further in vitro and in vivo experiments. [less ▲]

Purpose: The purpose of this work is to study the peptide encapsulation into PEGylated liposomes. Two formulations (SPC:CHOL:mPEG-750-DSPE (47:47:6) or SPC:CHOL:mPEG-2000-DSPE (47:47:6)) were investigated ... [more ▼]

Purpose: The purpose of this work is to study the peptide encapsulation into PEGylated liposomes. Two formulations (SPC:CHOL:mPEG-750-DSPE (47:47:6) or SPC:CHOL:mPEG-2000-DSPE (47:47:6)) were investigated. Methods: Blank SUV liposomes were prepared by the lipid film hydration and the encapsulation was achieved by applying freeze-thawing cycles. Because many factors may influence peptide entrapment (number of freeze-thawing cycles (NC), lipid concentration (LC), peptide concentration (PC), mixing time (MT) and liposome composition (COMP)), a design of experiment (DOE) was performed. Results: The screening permitted to identify two factors having a positive and significant influence on the encapsulation efficiencies (NC and LC) while the liposome composition had a relatively weak effect. For the second part of the DOE, the positive factors were optimized for liposomes containing mPEG2000. The obtained results revealed a theoretical optimum at 64.75±3.55% when 11 cycles were applied and for the following LC: 36.1mM SPC, 36.1mM CHOL and 4mM mPEG-2000-DSPE. Experimental results showed an encapsulation efficiency of 62.68±2.93%. Conclusion: The DOE led to significant improvement of encapsulation for liposomes containing mPEG2000. Thereafter, an optimization design for liposomes containing mPEG750 will be started. Acknowledgements: This work was supported by the Ministry of the Walloon Region. [less ▲]

Purpose: Print 3G is a peptidic antagonist of oncoprotein involved in breast cancer, containing 25 amino acids. The purpose of this work is to study the peptide encapsulation into PEGylated liposomes ... [more ▼]

Purpose: Print 3G is a peptidic antagonist of oncoprotein involved in breast cancer, containing 25 amino acids. The purpose of this work is to study the peptide encapsulation into PEGylated liposomes composed of SPC:CHOL:mPEG-2000-DSPE (47:47:6). Methods: Loaded unilamellar vesicles were prepared by hydration of lipid film method. Unfortunately, a loss of Print 3G was observed during the different steps of this manufacturing technique giving rise to encapsulation efficiencies (EE) close to 0 %. Thus, the freeze-thawing method was used to enhance the amount of Print 3G encapsulated into blank liposomes prepared using the above procedure. Because many factors may influence the peptide entrapment into the vesicles (number of freeze-thawing cycles (NC), lipid concentration (LC), peptide concentration (PC) and mixing time (MT)), a design of experiment (DOE) was performed (for the screening, a Plackett and Burman plan; for the optimization, a central composite design). EE were calculated in terms of quantity of peptide loaded in liposomes as a function of quantity operated (EEp) or quantity of lipids (EEl). Results: The EE obtained by the freeze-thawing method in standard conditions (Katanasaka, Ida et al., 2008; Maeda, Bharate et al., 2008), amounted to 26.20 ± 7.98 %, n=3 (EEp) and to 0.26 ± 0.074 % (EEl). Among the different considered parameters, the screening permitted to identify two factors having a positive and significant influence on the EE: NC and LC. Concerning the PC and MT, no influence was revealed. For the second part of the DOE, the positive factors were optimized and obtained results revealed a theoretical optimum at 64.75 ± 3.55 % when 11 freeze-thawing cycles were applied (NC) and for the following LC: 36.1 mM SPC, 36.1 mM CHOL and 4mM mPEG-2000-DSPE. The experimental results showed an encapsulation efficiency of 62.68 ± 2.93 %. Photon correlation spectroscopy and freeze-fracture electron microscopy were also realized to examine the liposome integrity, before and after the freeze-thawing cycles. Conclusion: Changing the manufacturing technique permitted a significant encapsulation of Print 3G into liposomes. The DOE led to a significant improvement of encapsulation efficiencies. References: (1) Katanasaka,Y., Ida,T., Asai,T., Maeda,N., Oku,N., 2008. Effective delivery of an angiogenesis inhibitor by neovessel-targeted liposomes. International Journal of Pharmaceutics, 360, 219-224. (2) Maeda,H., Bharate,G.Y., Daruwalla,J., 2008. Polymeric drugs for efficient tumor-targeted drug delivery based on EPR effect. Eur. J Pharm Biopharm. Acknowledgements: This work was supported by the Ministry of the Walloon Region. [less ▲]

Purpose: Print 3G is a peptidic antagonist of oncoprotein involved in breast cancer, containing 25 amino acids. The purpose of this work is to study the peptide encapsulation into PEGylated liposomes. Two ... [more ▼]

Purpose: Print 3G is a peptidic antagonist of oncoprotein involved in breast cancer, containing 25 amino acids. The purpose of this work is to study the peptide encapsulation into PEGylated liposomes. Two formulations composed of SPC:CHOL:mPEG-750-DSPE (47:47:6) or SPC:CHOL:mPEG-2000-DSPE (47:47:6) were investigated. Methods: Unilamellar vesicles containing either mPEG750 or mPEG2000 were prepared by hydration of lipid films method. Unfortunately, a loss of Print 3G was observed during the different steps of this manufacturing technique giving rise to encapsulation efficiencies close to 0 %. Thus, the freeze-thawing method was used to enhance the amount of Print 3G encapsulated into blank liposomes prepared using the above procedure. Because many factors may influence the peptide entrapment into the vesicles (number of freeze-thawing cycles, lipid concentration, peptide concentration, mixing time and liposome composition), a design of experiment was performed (for the screening, a Plackett and Burman plan; for the optimization, a central composite design). Results: The encapsulation efficiencies obtained by the freeze-thawing method in standard conditions, varied between 17.26 ± 0.46 % (n=3) for liposomes containing mPEG750 and 26.20 ± 7.98 % (n=3) for those comprising mPEG2000. Among the different considered factors, the screening permitted to identify two factors having a positive and significant influence on the encapsulation efficiencies: the number of freeze-thawing cycles and the lipid concentration, while the presence of mPEG2000 or mPEG750 had a relatively weak effect on the encapsulation. Concerning the peptide concentration and the mixing time, no influence was revealed. For the second part of the DOE, the positive factors were optimized for the liposomes containing mPEG2000. The obtained results for liposomes containing mPEG2000 revealed a theoretical optimum at 64.75 ± 3.55 % when 11 freeze-thawing cycles were applied and for the following lipid concentrations: 36.1 mM SPC, 36.1 mM CHOL and 4mM mPEG-2000-DSPE. The experimental results showed an encapsulation efficiency of 62.68 ± 2.93 %. Conclusion: Changing the manufacturing technique permitted a significant encapsulation of Print 3G into liposomes. The DOE led to a significant improvement of encapsulation efficiencies for the liposomes containing mPEG2000. Thereafter, an optimization design for liposomes containing mPEG750 will be started. Acknowledgements: This work was supported by the Ministry of the Walloon Region. [less ▲]